Stain coated slides for differentially staining blood

ABSTRACT

SLIDES ARE PRESTAINED WITH A HOMOGENEOUS MIXTURE OF METHYLENE BLUE NN AND CRESYL VIOLET ACETATE IN A DRY TRANSPARENT FILM. A DROP OF BLOOD SMEARED ON THE SLIDE WITH A COVER GLASS SHOWS RETICULOXYTES, PLATLETS, AND WHITE BLOOD CELLS (NEUTROPHILIS, EOSINOPHILS, BASOPHILS, LYMPHOCYTES, MONOCYTES) SEPARATELY IDENTIFIABLE AFTER APPROXIMATELY FIVE MINUTES STAINING AT ROOM TEMPERATURE.

United States Patent 3,796,594 STAIN COATED SLIDES FOR DIFFERENTIALLY STAINING BLOOD Frederick W. Thomae, Jr., King of Prussia, and John Geating, Abington, Pa., assignors to General Electric Company No Drawing. Continuation-impart of abandoned apphcation Ser. No. 85,817, Oct. 30, 1970. This application Feb. 1, 1972, Ser. No. 222,654

Int. Cl. C03c 17/00; G01n 1/30, 33/16 US. Cl. 117-124 D ABSTRACT OF THE DISCLOSURE Slides are prestained with a homogeneous mixture of methylene blue NN and cresyl 'violet acetate in a dry transparent film. A drop of blood smeared on the slide with a cover glass shows reticulocytes, platelets, and 'white blood cells (neutrophils, eosinophils, basophils, lymphocytes, monocytes) separately identifiable after approximately five minutes staining at room temperature.

CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of our copending prior application Ser. No. 85,817, filed Oct. 30, 1970, now abandoned.

BACKGROUND OF THE INVENTION (1) Field of the invention This invention pertains to biological cell identification by staining.

(2) Description of the prior art It is desirable to identify separately, in a blood sample, the reticulum of young viable erythrocytes, platelets, and the various components and constituents of the white blood cell total, such as eosinophils, cytoplasm, cytoplasmic granules, nuclei, and mitochondria. This has, in general, been accomplished by the application of a variety of stains to separate samples for determination of different components of interest. In general, the stains have been applied in solution, although some limited success has been reported using dried film of stain.

Basic vital dyes stain unfixed reticulocytes specifically; when a blood sample is fixed with methyl alcohol, the basic staining is diffuse. Platelet identification and white blood corpuscle differentiation are not achieved.

Sabin has described the use of a dried film of neutral red mixed with Janus green B, upon a slide to which a small amount of blood is added. The neutrophilic, basophilic, and eosinophilic granules are differentiated somewhat by the pH sensitivity of neutral red; mitochondria are counterstained by the Janus green B; and white blood corpuscles are differentiated by staining of cytoplasmic granules, size, and cell mobility-which requires that the cells must be kept alive, despite the moderate toxicity of the stains. Recent tests of this method of which we are cognizant did not produce satifactory results, possibly reflecting the fact that standards of adequacy have changed since the original Sabin report in 1923. The Sabin technique, as described in Approved Laboratory Technic, Kolmer, Spaulding, and Robinson, fifth edition, Appleton-Century-Crofts, Inc., New York City, N.Y., 1951, on pages 80 and 81 was followed exactly. Only extremely slight staining, inadequate for normally rapid differentiation of the blood cells, was observed. The relative proportions of neutral red and Janus green were then altered in both directions from those specified, but without any improvement in staining. It is considered possible that improvements in dye manufacture which Claims have presumably occurred in the nearly fifty years since Sabins work have caused modern dyes to lack unknown impurities which produced in the original Sabin preparations results not obtainable with modern stains, even though they bear the same designations.

In other prior art methods, brilliant cresyl blue is used to stain reticulocytes; but the white blood corpuscles are not differentiated, and the nuclei stain poorly, and the cytoplasmic staining is irregular.

Reticulocytes may be stained in a mixture of blood and methylene blue NN in saline solution spread and dried on a slide; but platelets are somewhat obscured by precipitate formation, and in the white blood corpuscles only the cytoplasmic granules are stained.

To summarize, none of the prior art has been found which permits separate identification of reticula, platelets, and leukocyte differentiation including staining of the nuclei, and differentiation of cytoplasmic granules and eosinophils, in a single specimen.

SUMMARY OF THE INVENTION A slide is coated with a mixture of methylene blue NN and cresyl violet acetate in absolute methanol, and dried rapidly. A drop of blood is applied to the stained dry surface, and spread by the weight of a cover glass. After five minutes of staining, reticulocytes, platelets, neutrophils (with distinction of cytoplasm and nuclei), eosinophils (with distinction of cytoplasma and nuclei), and basophils (with distinction of containing granules) are all separately identifiable. The slides may be prepared and stored dry until needed for use.

DESCRIPTION OF THE PREFERRED EMBODIMENT A solution of 2.0 grams of methylene blue NN (Color Index basic blue 24) in 25 milliliters of absolute methyl alcohol is prepared, and allowed to stand over night, i.e. from 16 to 24 hours. It is then filtered to remove any undissolved residue.

A solution of 1.0 gram of cresyl violet acetate in 25 milliliters of absolute methyl alcohol is prepared, and allowed to stand over night, i.e. from 16 to 24 hours. It is then filtered to remove any undissolved residue.

In a test tube 1.0 milliliter of methylene blue N solution described above is placed, 10 to 15 drops (0.13 to 0.2 ml.) of cresyl violet acetate solution described above is added to it, and mixed by swirling. This amount'sufiices to prepare about 40 slides.

A drop of the dye mixture is placed at the end of a clean microscope slide. Another glass slide is applied vertically on the drop and drawn broadside across the surface of the first slide. Capillary action between slide and liquid will insure even distribution of the dye. It is particularly important that the side be free of grease or fat and of particles such as dust. The slide is then immediately dried by waving in air, or by a blast of clean dry gas, such as air. The slide is ready for use immediately upon drying; but it may be stored for future use at low humidity. High giimidity has been found to cause deformation of the dye To use the slide, a sample site, eg finger, is sterilized conventionally with 70 percent methyl alcohol, and punctured with a blood lancet. The first drop of blood is wiped off. The next drop is applied directly to the prestained surface of the slide.

A cover slip is immediately placed over the small drop of blood. The weight of the cover slip may suflice to spread the drop uniformly over the slide; if it does not, slight pressure on the cover slip (conveniently with the clean end of a capillary tube) will cause it to spread thus.

After five minutes at room temperature, staining sufii cient for observation will be complete. The covered slide is then observed under a high power microscope, prefer-v ably with an oil immersion objective.

The staining characteristics differ from those of the prior art Wrights stain. The staining characteristics of the slide coating of our. invention are the following:

Neutrophils contain a finely granulated cytoplasm which stains a light purple in the fresh preparation and fades to a greenish tint in the older smear. Nuclei stain a bright purple.

Eosinophils exhibit a bright purple nucleus and a cytoplasm packed with large uniform orange-staining granules which fade to a greenish orange when the preparation is about twelve hours old. Basophils contain smaller number of uniform-size granules than eosinophils, which stain a dark purple and often completely obscure the nucleus. With focusing, an orange tinge can be observed in the granules found at the edge of the cell, an'effective identifying feature.

Lymphocytes develop a purple staining nucleus surrounded by a lighter purple cytoplasm which may become as dark as the nucleus as the preparation grows old.

Monocytes have an afiinityfor the stain similar to lymphocytes. Distinguishing features are the larger size and the greater amount of cytoplasm of the monocytes. It has been found that the differentiation is readily learned.

Platelets stain a pink color. A platelet estimation is best performed as soon after the staining period as possible because of an accumulation of debris with increasing time.

Reticulocytes contain a reddish-purple network but do not fade even after the red cells become distorted. Nevertheless, the reticulocyte count should also be made within 4 to 5 hours after the staining period because of the fading of some of the red blood corpuscles when they die.

References to the prior art are:

Brecher, G. 1949. New Methylene Blue as a Reticulocyte Stain, American Journal of Clinical Pathology, 19:895-6.

Conn, H. J. -(ed.), Biological Stains, Seventh Ed., Williams and Wilkins Company, Baltimore, Md., 1961.

Cunningham, J. H. 1920. A Method for Permanent Staining of Reticulated Red Blood Cells. Archives of Internal Medicine, 26:405-9.

Dacie, J. V. and Lewis, S. M. 1963. Practical'Haematology, 3rd Edition, pages 28-29.

Doan, C. A. and Ralph, P. H., In, McClungs I-Iandbook of Microscopical Technique, 3rd Edition, 1950, pages 571-585.

Frankel, S. and Reitman, S. .(eds.) Sonnerwirth, A. C. (asst. ed.) Gradwohls Clinical Laboratory Methods and Diagnosis; Sixth Edition, volume 2, C. V. Mosby Com pany, St. Louis, 1963, pages 1132-1134.

Spiridonovitch," R. 1924. Vital Staining of White Blood Cells with Cresylecht Violet. Anatomical Record, 27:367-373. M

Watson, C. I.' and Clark 1937. Occurrence of Protoporphyrine in Reticulocytes'. Proceedings of the Society of Experimental Biology and Medicine, 36:65-69.

4 Hepler, O. E., Manual of Clinical Laboratory Methods, Fourth Edition, Charles C. Thomas, Springfield, Ill., 1965,

. Hematology.

The possibility of using prestained slides, of reducing the staining time (and markedly simplifying the preparation procedure), and of making a multiple determination in a single slide, instead of employing several preparations for different determinations, are all marked advantages of our invention.

The proportion of methylene blue NN in the coating may range from approximately 91% to 94%, and that of cresyl violet acetate from approximately6% to 9% Moderate departures from these proportions are, .of course, permissible, and may be desirable if it is desired to accentuate particular constituent staining. Since it is the provision of the combination of stains to the blood sample which is essential to our invention, it is not necessary that the film of dried stains be applied to the slide; it is equally feasible to provide the dried dye film on a transparent film, which is applied over a drop of blood on a clean slide. Such prestained transparent films may be packaged in individual sealed envelopes, which would automatically provide a suitably dry atmosphere for storing the dye film, and would, of course, markedly simplify the problems of packaging for shipment, and of storage. By relieving the clinical laboratory of the handling of fluid dyes, and relegating the entire stain preparation to a factory,-the use of our invention minimizes the burden upon the clinical technician. Also, the elimination of various intermediate steps of the prior art not only reduces the time consumed in a determination but tends to a higher degree of uniformity in preparations.

We claim:

1. A microscopic slide having coated thereon a mixture of stains for differentially staining blood, said mixture consisting of approximately 91% to 94% by weight of methylene blue NN and 6% to 9% by weight of cresyl violet acetate.

2. A method of differentially staining a blood sample which comprises (a) drying on a microscopic slide an alcoholic solution containing approximately 91% to 94% by weight of methylene blue NN and 6% to 9% by weight of cresyl violet acetate, and

(b) applying the blood sample to said stain on said slide whereby the blood cell components become differentially stained.

References Cited Sabin: Bull. The Johns Hopkins Hospital, vol. 34, 1923, pp. 277-288.

, Brecher: AJCP, vol. 19, 1949, pp. 895-6.

ALBERT T. MEYERS, Primary Examiner A. P. FAGELSON, Assistant Examiner US. Cl. X.R. 117-3; 424-3 

